Serial monitoring of circulating tumour DNA on clinical outcome in myelodysplastic syndromes and acute myeloid leukaemia

Dear Editor, Diagnostic bone marrow (BM) DNA andmatched plasmaderived circulating tumour DNA (ctDNA) demonstrated excellent correlations in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML),1–4 and dynamic ctDNA monitoring contributes to predict relapse in MDS and AML patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT).5 However, few researches are reported on the application of ctDNAdynamicmonitoring assessments during thewhole disease course in MDS and AML.1 Here, we assessed the feasibility and utility of ctDNA as a novel and minimally invasive biomarker based on targeted next-generation sequencing (NGS) to monitor treatment outcome, track clonal evolution and predict survival. To the best of our knowledge, we firstly demonstrated the application of ctDNA concentration as an effective prognostic biomarker in MDS and AML. The details of clinical data and methods are shown in Tables S1–S3. Thirty-five adult patients were involved in our study. Twenty-seven patients had preand posttreatment plasma-derived ctDNA, and paired baseline BM DNA NGS assessments were included for both concordance analysis and dynamic ctDNA analysis. Four patients with only baseline BM DNA and plasma-derived ctDNA NGS assessments were used for concordance analysis. Three patients with only preand post-treatment ctDNA assessments and one patient with only post-treatment ctDNAassessmentswere used for dynamic ctDNAanalysis (Figure 1). A total of 46 mutated genes with 135 mutations were detected in BM DNA and plasma-derived ctDNA, involving 42 genes with 100 mutations (74.1%) detected both in BM DNA and plasma-derived ctDNA (Figure 2A, Figure S1). Among the 100 concordant mutations, the concordance of variant allele frequencies (VAFs) assessment based on plasma and BM was high (R = .854 p < .001)


Serial monitoring of circulating tumour DNA on clinical outcome in myelodysplastic syndromes and acute myeloid leukaemia
Dear Editor, Diagnostic bone marrow (BM) DNA and matched plasmaderived circulating tumour DNA (ctDNA) demonstrated excellent correlations in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), [1][2][3][4] and dynamic ctDNA monitoring contributes to predict relapse in MDS and AML patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). 5 However, few researches are reported on the application of ctDNA dynamic monitoring assessments during the whole disease course in MDS and AML. 1 Here, we assessed the feasibility and utility of ctDNA as a novel and minimally invasive biomarker based on targeted next-generation sequencing (NGS) to monitor treatment outcome, track clonal evolution and predict survival. To the best of our knowledge, we firstly demonstrated the application of ctDNA concentration as an effective prognostic biomarker in MDS and AML.
The details of clinical data and methods are shown in Tables S1-S3. Thirty-five adult patients were involved in our study. Twenty-seven patients had pre-and posttreatment plasma-derived ctDNA, and paired baseline BM DNA NGS assessments were included for both concordance analysis and dynamic ctDNA analysis. Four patients with only baseline BM DNA and plasma-derived ctDNA NGS assessments were used for concordance analysis. Three patients with only pre-and post-treatment ctDNA assessments and one patient with only post-treatment ctDNA assessments were used for dynamic ctDNA analysis ( Figure 1). A total of 46 mutated genes with 135 mutations were detected in BM DNA and plasma-derived ctDNA, involving 42 genes with 100 mutations (74.1%) detected both in BM DNA and plasma-derived ctDNA (Figure 2A, Figure  S1). Among the 100 concordant mutations, the concordance of variant allele frequencies (VAFs) assessment based on plasma and BM was high (R = . ( Figure 2B). Comparing pre-treatment mean ctDNA concentrations with blasts in BM at baseline measured by cytological analysis, we found pre-treatment mean ctDNA concentrations were strongly associated with BM blasts at baseline (R = .618, p < .001) ( Figure 2C), indicating mean ctDNA concentration were related to tumour burden. We also reached a moderate correlation between pre-treatment mean ctDNA VAF and BM blasts (R = .533, p = .001) ( Figure S2).
As expected, post-treatment ctDNA positivity was associated with both shorter progression-free survival (PFS, median PFS, 5.6 vs. not reached [NR] months, p < .001) ( Figure 3A) and overall survival (OS, median OS, 11.0 vs. NR months, p < .001) ( Figure 3B). In the meantime, increased mean ctDNA VAF (defined as increasing posttreatment compared with pre-treatment ctDNA VAF) predicted decreased PFS (median PFS, 2.8 vs. 25.0 months,    Figure 3D). Furthermore, we discovered patients with increased mean ctDNA concentration have poorer PFS (median PFS, 2.8 vs. NR months, p < .001) ( Figure 3E) and OS (median OS, 7.9 vs. NR months, p < .001) ( Figure 3F). However, the significant prognostic impact of pre-treatment ctDNA status on PFS and OS with mean ctDNA VAF ( Figure S4A,B) or mean ctDNA concentration ( Figure S4C and D) was not discovered, indicating pre-treatment ctDNA status was not a good biomarker to stratify the prognosis. To investigate the prognostic differences between mean ctDNA VAF and mean ctDNA concentration change from post-to pre-treatment, we compared the discrimination ability of the prognostic model  Figure 3G). Similarity was also discovered in OS analysis (C-index, .70 vs. .67; AUC, .78 vs. .72) ( Figure 3H).
Next, we tried to explore clonal evolution patterns in refractory or relapsed MDS and AML. We observed two patterns of progression from MDS to secondary AML (sAML). Patient 1 (P1) showed a linear pattern, where successive clones occurred within the previous parental clone. The acquisition of FLT3 mutation occurred during disease relapse and subsequently transformation to AML ( Figure 4A). P7 and P8 showed a branched pattern ( Figure 4B, Figure S5A, respectively). For P7, the original clone harboring PTPN11 mutation was suppressed while a new clone carrying FLT3-ITD mutation expanded, leading to AML transformation. However, we only discovered one linear pattern in refractory or relapsed AML. Three representative patients (P11, P26 and P35) achieved CR and relapsed within 6−7 months ( Figure 4C, Figure S5B,C, respectively). The founding clone in the primary tumour in P11 contained mutations in WT1, GATA2 and RAD21 that were all recurrent at relapse. A new subclone occurred and evolved to become the dominant clone at relapse by acquiring additional SETD2 mutations. Compared with flow cytometry measurable residue disease results, we found that ctDNA molecular residue detection was earlier about 4 months.
In summary, our study revealed that serial plasmaderived ctDNA assessments can reflect treatment response, survival, and clonal evolution in adult MDS and AML as a minimally invasive method, which warrant the prospective use of ctDNA as a biomarker in disease monitoring.

A C K N O W L E D G E M E N T S
This work was supported by the National Natural Science Foundation of China (grant number: 81800121) and Zhejiang Medical Association Clinical Research Fund project (grant number: 2017ZYC-A14). The authors also thank all the patients who participated in this study.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare that they have no competing interests.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data used in the present study are available from the corresponding author upon reasonable request.